Important Pathogenic Bacteria During Child Growth

Evita Mayasari, dr., MKes.

Microbiology Department Medical Faculty, University of Sumatera Utara

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First isolated by Pfeiffer during the influenza pandemic of 1890

Family: Pasteurellaceae Small Gram (-) coccobacilli or filamentous

rods, hence the descriptive term pleomorphic. Polysaccharide capsule (+) : consists of a

repeating polymer of 5 carbon sugar units, ribose, and ribitol phosphate.

Envelope includes lipo-oligosaccharide (LOS) and outer-membrane proteins (OMP).

Pili or fimbriae presence appears to be variable

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Case: Upper Airway Problems in Children: Acute Epiglottitis

Grow on chocolate agar, where they have a glistening, semitransparent appearance.

Identified by the requirement for X (hemin or other porphyrins) and V (coenzyme NAD) factors for growth on blood agar.

A more sensitive test for the X factor requirement is to test the ability of H. influenzae to convert delta aminolevulinic acid to porphyrin.

Other tests such as the production of indole from tryptophan and detection of β-galactosidase (ONPG test) activity are also useful in discriminating H. influenzae from other Haemophilus species.

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Tissues infected by type b and nontypable strains of H. influenzae

The major virulence factor: type b polysaccharide capsule allows them to resist phagocytosis and complement-mediated lysis in the nonimmune host.

Encapsulated organisms can penetrate the epithelium of the nasopharynx and invade the blood capillaries directly Bacteremia spread to CNS and distant sites such as bones and joints

Attachment to respiratory epithelial cells is mediated by pili and other adhesins.

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Respiratory route (person to person) Close contact with patients suffering H.

Influenzae infection presents a definite risk for nonimmune persons and nonimmune children <4 years old.

Prophylaxis with Rifampin is recommended for such children.

Vaccination with type b polysaccharide (in the form of Hib conjugate vaccines) is effective in preventing infection.

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Blood CSF (cerebrospinal fluid) Prompt transport of those samples is mandatory to

ensure the fastest possible diagnosis and survival of m.o. in the sample

Other specimens: aspirated synovial fluid, pericardial fluid, pleural fluid, pus, sputum, nasopharyngeal or throat swabs, purulent discharge from infected eyes.

For samples taken from respiratory tract: avoid contamination with commensals throat swabs must be collected from the pharynx

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Samples must be transported to the lab. in a suitable transport medium (thioglycolate-hemin-based)

Or samples should be spread directly onto appropriate agar media whenever possible (esp. conjunctival specimens)

The viability of most haemophilus is readily lost as a result of drying out, they do not survive more than a few days in clinical samples.

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1906: first isolated in pure culture by Bordet and Gengou

Tiny (0.5 to 1.0 m), Gram-negative coccobacillus morphologically much like Haemophilus.

The surface exhibits a rod-like protein called the filamentous hemagglutinin (Fha) because of its ability to bind to and agglutinate erythrocytes.

Contains surface pili Capsule includes a protein called pertactin. Strict aerobe, nonmotile, non–spore forming Extracellular products: Pertussis toxin (PT),

adenylate cyclase toxin, tracheal cytotoxin

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Bordetella pertussis, the agent of pertussis or whooping cough. Gram stain. (CDC)

Culture media enriched with blood: Bordet-Gengou, charcoal-horseblood agar (Regan-Lowe)

Incubated in moist environment, slow growth (3 to 7 days)

Identified by immunofluorescence staining Forms acid but no gas from glucose and lactose Hemolysis of blood (in medium) is associated

with virulent B. pertussis Bordetella DNA can also be detected by PCR.

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"mercury drop" colonies on Bordet-Gengou is likely to be B. pertussis

Mortimer & Jones, 1979: in USA, ±5 of every 1,000 infants born alive died of the disease before their fifth birthdays

After the introduction of immunization (1940), the incidence dropped from 250,000 cases a year to below 1/100,000 population (in US)

Now well controlled in the US and other developed countries

MANIFESTATIONS: incubation period (7-10 days) stages: (1) catarrhal; rhinorrhea that persists for 1-2 weeks, malaise, fever, sneezing, and anorexia may also be present (2) paroxysmal; persistent cough up to 50 times a day for 2-4 weeks. Whoop, vomiting, apnea (infants) (3) convalescent; frequency and severity (3-4 weeks)

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The agent of whooping cough is transmitted primarily via droplets.

1. Introduced into the respiratory tract. It has a tropism for ciliated bronchial epithelium attaching to the cilia themselves. This adherence is mediated by Fha, pili, pertactin, and the binding subunits of PT.

2. Once attached, the bacteria immobilize the cilia then progressively destroyed the ciliated cellsThis local injury is caused primarily by the action of the tracheal cytotoxin.

3. This produces an epithelium devoid of the ciliary blanket, which moves foreign matter away from the lower airways.

4. Persistent coughing is the clinical correlate of this deficit.

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Synergy between pertussis toxin and the filamentous hemagglutinin in binding to ciliated respiratory epithelial cells.

Nasopharyngeal (NP) aspirates Posterior NP swabs If properly collected, those specimens contain the

ciliated respiratory epithelial cells for which B. pertussis exhibit tropism

Inferior specimens: throat swabs, but may be suitable for PCR diagnosis of B. pertussis infection

Recommended: collect two NP swab specimens, one through each nostril

Specimens are then directly plated or placed in suitable transport media: Casamino Acids or Amies with charcoal

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Slim, strongly acid-alcohol-fast rod 2-4 m (length) and 0.2-0.5 m (width)

Nonmotile, obligate aerobes,do not form spores The cell wall contains peptidoglycan similar to

Gram(+) organisms, except that it contains N-glycolylmuramic,rather than N-acetylmuramic, acid.

Attached to peptidoglycan are: polysaccharides, proteins, and lipids; long-chain fatty acids called mycolic acids (make up >60% of the total cell wall mass)

Enveloped

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Grows at 37°C but not at room temperature Growth is enhanced by 5 to 10% CO2

Requires enriched or complex media for primary growth: Lowenstein–Jensen

The dry, rough, buff-colored colonies usually appear after 3-6 weeks of incubation.

Due to its hydrophobic lipid surface, M. tuberculosis is unusually resistant to drying,to common disinfectants, and to acids and alkalis.

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Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium. CDC.

Sensitive to heat, including pasteurization, and individual organisms in droplet nuclei are susceptible to inactivation by UV light.

Staining characteristic: acid-fast (Ziehl-Neelsen stain method). Once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds.

The bacterium is a facultative intracellular parasite, usually of macrophages, and has a slow generation time, 15-20 hours

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Mycobacterium tuberculosis. Acid-fast stain. CDC.

There are 5 stages of the disease (tuberculosis)

Stage 1 Droplet nuclei are inhaled. One droplet nuclei contains no > 3 bacilli. Droplets are so small that they can remain

air-borne for extended periods of time. Droplets are generated by during talking,

singing, coughing, and sneezing.

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Spread of droplet nuclei from one individual to another. After droplet nuclei are inhaled, the bacteria are nonspecifically taken up by alveolar macrophages. However, the macrophages are not activated and are unable to destroy the intracellular organisms.

CDC.

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Stage 2 Begins 7-21 days after initial infection. MTB multiplies unrestricted within

unactivated macrophages until the macrophages burst.

Other macrophages begin to extravasate from peripheral blood.

These macrophages also phagocytose MTB, but they are also unactivated and hence can not destroy the bacteria.

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Stage 3 Lymphocytes begin to infiltrate recognize MTB

antigen T-cell activation and the liberation of cytokines including gamma interferon (IFN) activation of macrophages

These activated macrophages are now capable of destroying MTB.

Positive tuberculin reaction Tubercle formation begins. The center is

characterized by "caseation necrosis“= takes on a semi-solid or "cheesy" consistency.

MTB cannot multiply within tubercles because of the low pH and anoxic environment. MTB can, however, persist within these tubercles for extended periods.

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Stage 4 MTB uses the unactivated macrophages to

replicate, and hence, the tubercle grows. The growing tubercle may invade a

bronchusspread to other parts of the lung The tubercle may invade an artery or other

blood supply line hematogenous spread extrapulmonary tuberculosis, known as milliary tuberculosis usually involve the genitourinary system, bones, joints, lymph nodes and peritoneum.

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Stage 5 For unknown reasons, the caseous centers

of the tubercles liquefy. This liquid is very conducive to MTB

growth, and the organism begins to rapidly multiply extracellularly.

After time, the large antigen load causes the walls of nearby bronchi to become necrotic and rupture. This results in cavity formation. This also allows MTB to spread to other parts of the lung.

Note: only a smaller percentage of MTB infections progress to an advanced stage

Slim, Gram-positive rod, forms a typical round terminal spore that gives the organism a drumstick appearance

The spores remain viable in soil or culture for many years. It is resistant to most disinfectants and withstands boiling for several minutes.

Flagellate and motile, strict anaerobic The most important product : neurotoxic

exotoxin, tetanospasmin or tetanus toxin Found in soils and in the intestinal tracts and

feces of various animals

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C. tetani Gram stain. 

Tetanus bacilli multiply locally and neither damage nor invade adjacent tissues, but synthesizes the tetanus toxin

The tetanus toxin initially binds to peripheral nerve terminals transported within the axon and across synaptic junctions until it reaches the CNS rapidly fixed to gangliosides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis block the release of inhibitory neurotransmitters (glycine and γ-amino butyric acid), which is required to check the nervous impulse.

If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus.

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Pathogenesis of tetanus caused by C tetani

Well growth on CDC anaerobe blood agar and phenylethyl alcohol blood agar (PEA) (1-2 days)

Non-selective media: Brucella agar + 5% sheep blood, columbia agar, brain heart infusion agar

Isolated colonies: subcultured to chopped-meat medium, incubated overnight

Spore selection techniques: alcohol treatment, heat treatment

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Preliminary identification: Gram stain, colony morphology: swarming, indole reaction: variable, spore location: terminal, drumstick shaped.

Definitive identification: PRAS Biochemical Inoculation, Gelatin Hydrolysis : (+), Esculin Hydrolysis: (-), Carbohydrate Fermentation

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Aerobic and facultative Gram-positive rods. 0.5-1 m in diameter Irregular swellings at one end: “club shaped”

appearance Granules are irregularly distributed within the

rods aniline dyes Produce diphtheria toxin (DT) inactivates

elongation factor EF-2 arrest of protein synthesis necrotizing and neurotoxic effect

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C. diphtheriae cells stained by Albert's technique . Note the characteristic "Chinese-letter" arrangement of cells.

Susceptible persons may acquire toxigenic diphtheria bacilli in the nasopharynx.

The organism produces DT that inhibits cellular protein synthesis and is responsible for local tissue destruction and membrane formation.

The toxin produced at the site of the membrane is absorbed into the bloodstream and then distributed to the tissues of the bodyresponsible for the major complications of myocarditis and neuritis and can also cause low platelet counts (thrombocytopenia) and protein in the urine (proteinuria).

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Specimen: dacron swabs from the nose, throat: -Obtained before administered of Antimicrobial drugs -Collected from beneath any visible membrane

The swab then placed in semisolid transport media (e.g.: Amies)

Smears stained with Gram stain On blood agar: colonies are small, granular, gray,

irregular edges, may have small zone of hemolysis On potassium tellurite agar: brown-black halo

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Modification elek method described by WHO

PCR- based methods: detect the DT gene (tax)

ELISA : detect DT from clinical C. diphtheriae isolates

Immunochromographic strip assay: detect DT in hours and highly sensitive

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